Identification And Function Study Of The Key Terpene Synthase(TPS) In The DMNT Synthesis Pathway Of Alternanthera Philoxeroides

Alligator weed,Alternanthera philoxeroides,is a perennial herb of the Amaranthaceae Alternanthera,native to South America.It has spread widely to wet land from 32 degrees north latitude to 38 degrees south latitude,causing serious economic and ecological problems.Agasicles hygrophila(Coleoptera: Chrysomelidae),hereinafter referred to as "flea beetle",is an excellent natural enemy that is introduced from South America and for the prevention and control of A.philoxeroides.Due to the high host specificity of A.hygrophila to its host A.philoxeroides,they become an excellent model for studying the interaction between monophagous insects and their hosts.Preliminary studies have shown that host volatile DMNT is a significant attractant of A.hygrophila,allowing A.hygrophila to discriminate host from non-host plants.DMNT is therefore considered to be an important volatile cue for A.hygrophila to orient,locate,and feed on A.philoxeroides.As an important key enzyme in the biosynthesis of DMNT,terpene synthase is an important point in understanding the monophagous insect selection,hosts adaptation,and the defense of host plants.In this study,the transcriptome data of the mixed samples of A.philoxeroides was used to screen and identify the terpene synthase genes related to the DMNT biosynthesis.The function of ApTPSs in the interaction between the monophagous A.hygrophila and its host A.philoxeroides was clarified.The interaction mechanism between the monophagous A.hygrophila and the host A.philoxeroides was clarified from the perspective of the metabolism of plant terpenoid volatiles.The main findings of this paper are as follows:1.First,by investigating the short-term behaviors of A.hygrophila feeding on A.philoxeroides leaves,the time delineation of contact and feeding behaviors was conducted.Then the relative expression levels of the 7 candidate ApTPSs fragments in the leaves after being contact for 5 min,feeding for 1 h,and mechanically damaged,and the expression changes over time after damage were analyzed using qPCR.It was found that the short-term contact of A.philoxeroides induced the up-regulated expression of ApTPS10,ApTPS12,ApTPS16 and ApTPS19;A.philoxeroides feeding strongly induced the up-regulated expression of 7 candidate ApTPSs.In the state of mechanical damage,the 7 candidate ApTPSs all showed relatively weak delayed up-regulation.Combining the DMNT volatilization under the induction of feeding and mechanical damage induced and the induced expression characteristics of ApTPSs,the possible ApTPSs related to DMNT biosynthesis were further screened.2.Four ApTPSs were assembled and cloned: ApTPS10,ApTPS15,ApTPS19 and ApTPS35.Bioinformatics analysis of the candidate ApTPSs found that they are all members of the TPS-g family,catalyze the formation of acyclic terpenes,and are in line with the characteristics of DMNT biosynthetic terpene synthase.The optimal induction conditions of each recombinant ApTPSs protein were screened using prokaryotic expression.Four ApTPSs catalyzed product components were determined in vitro using SPME and GC-MS,then the catalytic function of each ApTPSs was preliminarily determined.Among them,ApTPS15 is a bifunctional enzyme that catalyzes the formation of nerolidol,the precursor of DMNT,as well as the formation of linalool.ApTPS10,ApTPS19 and ApTPS35 all catalyze the formation of linalool.3.The tissue expression analysis of 4 ApTPSs revealed that ApTPS15 had the highest relative expression in A.philoxeroides leaves,while the relative expression of ApTPS19 showed little difference.The ApTPSs-EGFP fusion expression vectors was further constructed and the tobacco transient expression system was used to analyze the subcellular localization of ApTPSs.It was found that ApTPS10 is located in the plastid,ApTPS15,ApTPS19 and ApTPS15 are all located in the cytoplasm.ApTPS15 is in line with the subcellular localization of terpene synthase responsible for DMNT biosynthesis.4.In order to study the contribution of ApTPS15 in the interaction of A.philoxeroides and A.hygrophila,overexpression vectors of ApTPSs were constructed.An Arabidopsis line overexpressing ApTPS15 and an Arabidopsis line overexpressing ApTPS19 for the reverse control was obtained using the Arabidopsis genetic transformation system.Overexpressing ApTPS15 Arabidopsis leaves are smaller and curlier,and the expression level of ApTPS15 in the leaves is more than twice that of the wild type;the overexpression of ApTPS19 in Arabidopsis is not much different from the wild type,and the expression level of ApTPS15 can reach more than 8 times that of the wild type.Compared with wild-type Arabidopsis thaliana,A.hygrophila female adults showed a clear preference for Arabidopsis lines overexpressing ApTPS15,but no obvious preference for Arabidopsis lines overexpressing ApTPS19.The results indicate that the expression of ApTPS15 can affect the behavioral selection of A.hygrophila female adults.5.Finally,the pGR107-ApTPS15 RNAi vector was constructed and ApTPS15 in A.philoxeroides was induced silencing by VIGS technology.ApTPS15-silenced plants showed a decrease in the abundance of nerolidol,and some plants also appeared to increase the abundance of other terpenoid volatiles.The female adults of A.hygrophila were more likely to choose the control A.philoxeroides instead of the ApTPS15-silenced A.philoxeroides.This result further proved that ApTPS15 was the terpene synthase that regulates the biosynthesis of DMNT precursor,nerolidol.In summary,ApTPS15 is the terpene synthase gene that encodes the synthesis of nerolidol,the precursor of A.philoxeroides DMNT.Its expression changes can significantly change the content of nerolidol in the leaves of A.philoxeroides,and then affect the behavioral selection of female adults of A.philoxeroides natural enemy A.hygrophila.This result helps to understand the relationship between monophagous insects and host plants.